A new multichannel method quantitating TUNEL in detached photoreceptor nuclei.

Area of Science:

• Ocular research
• Cell death and apoptosis
• Biomedical imaging analysis

Background:

• TUNEL assay is a gold standard for apoptosis detection in ocular research.
• Existing quantification methods like ImageJ RA Toolkit and ImagePro lack nuclear counterstain verification.
• Accurate nuclear co-localization is crucial for reliable cell death assessment.

Purpose of the Study:

• To establish and validate a new ImageJ-based automated multichannel thresholding (MCT) method for quantifying nuclear co-localized TUNEL labeling.
• To compare the MCT method against established quantification techniques.
• To ensure accurate apoptosis detection by incorporating nuclear counterstain verification.

Main Methods:

• Developed an ImageJ-based automated multichannel thresholding (MCT) method.
• Induced experimental retinal detachment (RD) in murine eyes.
• Acquired cross-sectional images of TUNEL and DAPI counterstain.
• Classified images into 'typical' and 'hotspot' TUNEL regions.
• Compared MCT results with ImagePro and ImageJ RA Toolkit (standard and high settings) using Bland-Altman and linear regression analyses.

Main Results:

• The MCT method showed strong correlation with ImagePro (R²=0.8972 for typical, R²=0.9000 for hotspot).
• RA Toolkit showed variable correlation with MCT, often yielding significantly higher counts.
• MCT method demonstrated reliable quantification and avoided staining artifacts by confirming nuclear co-localization.

Conclusions:

• The automated multichannel thresholding (MCT) method provides a validated and accurate approach for quantifying nuclear co-localized TUNEL labeling.
• MCT offers an improvement over existing methods by incorporating nuclear counterstain verification, reducing potential artifacts.
• This new method is a valuable tool for ocular research, particularly in studying apoptosis in conditions like retinal detachment.